A. Field of the Invention
This invention pertains to the field of processing aloe plants and removing portions of said plant for processing same into compositions for topical and internal applications and compositions of matter comprising said portions of aloe and uses thereof.
B. Description of the Prior Art, and Other Information
Aloe vera is not a cactus plant, as widely believed, but rather a member of the lily family. There are about 360 species of aloe plants known. Harding, Aloes of the World: A Checklist, Index and Code, Excelsa 9: 57-94 (1979). They seem to thrive in hot, arid areas and are widely scattered from the Mediterranean Sea, Middle East, Africa, China, Japan, Mexico and the southern U.S.A. A few of the important species used for their medicinal properties are Aloe barbadensis Miller (aloe vera), A. arborescens, A. plicatilis, A. vahombe, A. saponaria, A. africana, A. ferox and Aloe perryi. Reynolds, Aloes of Tropical Africa and Madagascar, The Trustees, The Aloe Book Fund, Mbabane Swaziland. However, A. barbadensis Miller is generally recognized as the "true aloe" because of its wide use and, reportedly, most effective healing power, although in Japan, A. arborescens Miller traditionally has been used as a folk remedy for various ailments ranging from gastrointestinal disorders to athlete's foot.
Aloe vera is a perennial plant with turgid green leaves joined at the stem in a rosette pattern. The leaves of a mature plant may be more than 25 inches long with saw-like spikes along their margins.
Slicing the leaf transversely as shown in FIGS. 1 and 2 reveals the outer walls of the epidermis (3) covered with thick cuticles. Beneath the epidermis (3) is the mesophyll which is differentiated into chlorenchymal cells and thinner walled cells known as parenchyma. The parenchymal cells harbor a transparent mucilaginous jelly (1). The vascular bundles (2) with inner bundle sheath cells contain the yellow sap, which has laxative properties, and are sandwiched between the two major cells. Needle-shaped crystals of calcium oxalate, produced as a metabolic by-product in plant cells, are found mostly at the central portion of the leaf.
Aloe vera contains two major liquid sources, a yellow latex (exudate) and the clear gel (mucilage). The dried exudate of Aloe barbadensis Miller leaves is referred to as aloe. The commercial name is Curacao aloe. It is composed mainly of aloin, aloe-emodin and phenols. Bruce, South African Medical Journal, 41: 984 (1967); Morrow et al., Archives of Dermatology, 116: 1064-1065 (1980); Saleh et al., Corrosion Prevention & Control, 9-10 (1983); Mapp et al., Planta Medica, 18: 361-365 (1970); Rauwald, Archives Pharmazie, 315: 477-478 (1982). A number of phenolics, including anthraquinones and their glycosides, are known to be pharmaceutically active. Bruce, Excelsa 5: 57-68 (1975); Suga et al., Cosmetics and Toiletries, 98: 105-108 (1983).
The mucilaginous jelly from the parenchymal cells of the plant is referred to as Aloe vera gel. There are generally no anthraquinones to decompose and cause discoloration of the gel unless the gel is contaminated by an improper processing technique.
Aloe vera gel is about 98.5% water by weight. More than 60% of the total solid is made up of polysaccharides of carbohydrate origin. Organic acids and inorganic compounds, especially calcium oxalate, account for the remainder of the solid.
Whole leaves, exudates and fresh gels of Aloe plants have been used for a variety of human afflictions. Evidence of their use as a medicinal remedy can be traced to the Egyptians of 400 BC. Aloe vera was also used to embalm the dead, as well as to protect the embalmers from the death-causing agent. Other early civilizations used aloe vera for skin care, to relieve insect stings and bites, to treat scratches and ulcerated skin, to promote wound healing, to prevent hair loss and as a purgative. It was the traditional medicine of many cultures as an anthelmintic, cathartic and stomachic and was used inter alia for leprosy, burns and allergic conditions. Cole et al., Archives of Dermatology and Syphilology, 47: 250 (1943); Chopra et al., Glossary of Indian Medicinal Plants, Council of Scientific and Industrial Research, New Delhi; Ship, Journal of the American Medical Association, 238:1770(1977); Morton, Atlas of Medicinal Plants of Middle American Bahamas to Yucatan, Charles C. Thomas Publisher, 78-80 (1981); Diez-Martinez, La Zabila, Communicado N). 46 Sobre Recursos Bioticos Potenciales del Pais, INIREB, Mexico (1981); Dastur, Medicinal Plants of India and Pakistan; D. B. Taraporevala Sons & Co., Private Ltd., Bombay 16-17 (1962).
Aloe vera has enjoyed a long history of lay acceptance as possessing "curative" or "healing" qualities. Over the last few years, numerous books and articles meeting scientific standards have been written on Aloe vera. Organizations such as the Aloe Vera Council and recognized medical institutions, through publications and case-histories of physicians, veterinarians and other scientists, have given credence to the "aloe phenomenon". Aloe vera has been featured extensively in the field of dermatology, especially for treating radiation-caused skin conditions. Mackee, X-Rays and Radium in the Treatment of Diseases of the Skin, 3rd Ed., Lea and Febiger, Philadelphia, 319-320 (1938); Rovatti et al., Industrial Medicine and Surgery, 28: 364-368 (1959); Zawahry et al., Quotations From Medical Journals on Aloe Research, Ed. Max B. Skousen, Aloe Vera Research Institute, Cypress, Calif., 18-23 (1977); Cera et al., Journal of the American Animal Hospital Association, 18: 633-638 (1982). The body of scientific literature documenting medical applications in digestive problems, as a virucidal, bactericidal and fungicidal agent and in gynecological conditions is extensive and has been adequately reviewed by Grindlay and Reynolds (Journal of Ethnopharmacology, 16: 117-151 (1986)).
The importance of chemicals found in aloes is indicated by the fact that they have been listed in every known national pharmacopeia. U.S. Pharmacopeia, 20th Revision, The National Formulary, 15th Edition, United States Pharmacopeial Convention, Inc., Rockville, Md., Jul. 1, 1980. However, the U.S. Pharmacopeia describes the yellow sap drug portion of aloes but not the mucilage. The fresh unpreserved gel is about 98.5-99.2% water. The total solid that remains after the water has been removed ranges from 0.8 to 1.5%. The mucilage, sugars, fiber, proteins, ash, fats, aloin and resin are the major constituents of the solid. Robson et al., Journal of Burn Care Rehabilitation, 3: 157-163 (1982). Compositions that include enzymes, organic acids, inorganic salts, amino acids and alkaloids have been noted. Rowe et al., Journal of the American Pharmaceutical Association, 30: 262-266 (1941); Roboz et al., Journal of the American Chemical Society, 70: 3248-3249 (1948); Waller et al., Proceedings of Oklahoma Academy of Science, 58: 69-76 (1978). Depending on the way in which the leaves are processed, mucilage and sugars are the major components of the dehydrated gel. The sugars found are galactose, glucose, mannose, rhamnose, xylose and uronic acids. Although conflicting reports have been observed, the mucilage is mainly composed of mannan or glucomannan. Eberendu et al., The Chemical Characterization of Carrisyn.TM. (in preparation); Mandal et al., Carbohydrate Research, 87: 249-256 (1980b); Roboz et al., Journal of the American Chemical Society, 70: 3248-3249 (1948); Gowda et al., Carbohydrate Research, 72: 201-205 (1979); Segal et al., Lloydia, 31: 423 (1968).
Prior to this work, the controversy over the identity of the active substance(s) in Aloe vera had not been settled. It is therefore important to clearly distinguish between the components present in the gel and those found in the exudates. A larger part of the gel is a mucilage of mainly polysaccharide nature with minor amounts of various other compounds. It has been observed that in some of the activities observed there may be some synergistic action between the polysaccharide base and other components. Leung, Excelsa 8: 65-68 (1978); Henry, Cosmetics and Toiletries, 94: 42-50 (1979). For example, several workers report that the effective components for wound healing may be tannic acid (Freytag, pharmazie, 9:705 (1954)) and a kind of polysaccharide. Kameyama et al., Japanese Patent #7856995 (1979). Mackee, supra, noted that the gel, not the rind or the exudate, was responsible for the beneficial effects in the treatment of radiation burns, and he stressed the importance of using fresh leaves for effective treatment. Polysaccharides degrade with time, and certain molecular weight sizes may be necessary to elicit a specified pharmacological response. Goto et al., Gann, 63: 371-374 (1972).
There are many examples in the literature that polysaccharides can exhibit pharmacological and physiological activities without help from other components. G. Gialdroni-Grassi, International Archives of Allergy and Applied Immunology, 76 (Suppl. 1): 119-127 (1985); Ohno et al., Chemical and Pharmaceutical Bulletin, 33: 2564-2568 (1985); Leibovic et al., Chemico-Biological Interactions, 60: 191-200 (1986); Ukai et al., Chemical and Pharmaceutical Bulletin, 31: 741-744 (1983); Leibovici et al., Anticancer Research, 5: 553-558 (1985). One such example relates to development of atherosclerosis. Hyperlipidemia in the general population and especially in familial hypercholesterolemia is associated with coronary heart disease and death. In countries where dietary fiber intake is high, atherosclerosis appears to be uncommon. Trowell et al., Editors, Refined Carbohydrate Foods and Disease, London, Academic Press, 207-209 (1975). Pectin and guar are reported to lower cholesterol in normal and hyperlipidemic patients. Kay et al., American Journal of Clinical Nutrition, 30: 171-175 (1977). Locust bean gum, a polysaccharide composed of mannose and galactose, decreased the plasma lipoprotein cholesterol concentrations in both normal and familial hypercholesterolemic subjects. Zavoral et al., American Journal of Clinical Nutrition, 38: 285-294 (1983). Addition of guar gum to carbohydrate meals decreased the postprandial rise of glucose in both normal and diabetic subjects. Jenkins et al., Lancet, 2: 779-780 (1977). Kuhl et al., (Diabetes Care, 6 (2): 152-154 (1983)) demonstrated that guar gum exhibited glycemic control of pregnant insulin-dependent diabetic patients.
The anti-tumor activity of polysaccharides has been widely reported. Polysaccharides prepared from Lentinus cyathiformis are known to increase hosts' defense against tumors. Rethy et al., Annales Immunologiae Hungaricae, 21: 285-290 (1981). There are several reports that polysaccharides from mushroom, yeast or bacterial extracts can elicit a high degree of host defense activity against viral and tumor infestations. Chihara et al., 222: 687-688 (1969); Shwartzman, Proceedings of the Society for Experimental Biology and Medicine, 29: 737-741 (1932); Rethy, X. International Congress of Microbiology; Moscow, 642 (1966). Suzuki et al. (Journal of Pharmacobio-Dynamics, 7: 492-500 (1984) also reported anti-tumor activity of a polysaccharide fraction (GF-1) extracted from cultured fruiting bodies of a fungus, Grifoloa frondosa. This fraction showed equivalent, high levels of inhibiting activity when administered intraperitoneally (IP), intravenously (IV) and intratumorally (IT). However, oral administration (PO) was not effective. The GF-1 fraction also exhibited anti-tumor action against the solid form of Meth A fibrosarcoma and MM 46 carcinoma in mice. Lentinan, which is a 6-branched .beta.-(1-3)-linked glucan similar to GF-1, was ineffective against Meth A fibrosarcoma. Chihara, The antitumor polysaccharide Lentinan: an overview; "Manipulation of Host Defense Mechanisms"; ed. by Aoki et al., Excerpta Medica, North Holland, 1-16 (1981); Sasaki et al., Carbohydrate Research, 47: 99-104 (1976). Synthesized branched polysaccharides were reported to demonstrate activities against tumors. Matsuzaki et al., Makromol. Chem., 186: 449 (1985). Matsuzaki et al. (Makromol. Chem., 187: 325-331 (1986)) synthesized branched polysaccharides, which showed significant activities, from ivory nut mannan (B-(1-4)-D-mannopyranose) and B-(1-4)-linked glucomannan. A partially acetylated linear B-(1-3)-D-mannan extracted from fruit bodies of Dictyophoria indusiata Fisch, also exhibited anti-tumor activity. Hara et al., Carbohydrate Research, 111: 143-150. It appears that anti-tumor action depends on the type of polymer main chain and its degree of polymerization, because B-(1-3)-glucan-type polymers show higher anti-tumor activity than B-(1-4)-glucan and hemicellulosic polymers. Matsuzaki et al., Makromol. Chem., 187: 325-331 (1986). A carboxymethylated derivative of B-(1-3)-glucan obtained from bacterial culture filtrate caused severe cell loss from established sarcoma 180 tumors within 2 hours after the injection of the derivative. Baba et al., Journal of Immunopharmacology, 8: 569-572 (1986). The same author observed a compensatory increase in polymorphonuclear leukocytes due to injection of the substance. Incidentally, bestatin, a dipeptide known to possess immune-modulating and anti-tumor activity (Ishizuka et al., Journal of Antibiotics, 32: 642-652 (1980)), influenced neither the tumor yield nor the polymorphonuclear leukocyte count. Baba et al., supra.
There are numerous reports on the anti-tumor effect of sulfated polysaccharides, including heparin (Jolles et al., Acta Univ. Int. Cancer, 16: 682-685 (1960); Suemasu et al., Gann, 61: 125-130 (1970)), sulfated laminaran and dextran. Jolles et al., British Journal of Cancer, 17: 109-115 (1963).
Yamamoto et al. (Japanese Journal of Experimental Medicine, 54: 143-151 (1984)) reported enhancement of anti-tumor activity of a fucoidan fraction by further sulfation. The sulfated product demonstrated activity against L-1210 leukemia. The authors postulated that the mechanism of the anti-tumor action might be due partly to inhibition of invasive growth of L-1210 cells, as a result of electrostatic repulsion between the tumor cell and mesothelial cells. Yamamoto et al., supra. Polysaccharides with sulfate groups are also reported to be human T cell mitogens and murine polyclonal B cell activators. Sugawara et al., Microbiological Immunology, 28: 831-839 (1984). Generally, homopolysaccharides of high molecular weight with sulfate groups possess these properties. Dorries et al., European Journal of Immunology, 4: 230-233 (1974); Sugawara et al., Cell Immunology, 74: 162-171 (1982).
It has been reported that glucan extracted from the yeast Saccharomyces cerbisiae is a modulator of cellular and humoral immunity. Wooles et al., Science 142: 1078-1080 (1963). The polysaccharide also stimulated proliferation of murine pluripotent hematopoietic stem cells, granulocyte macrophage colony-forming cells and cells forming myeloid and erythroid colonies. Pospisil et al., Experientia, 38: 1232-1234 (1982); Burgaleta et al., Cancer Research, 37: 1739-1742 (1977). Maisin et al. (Radiation Research, 105: 276-281 (1986)) also reported that IV administration of a polysaccharide induced protection of murine hematopoietic stem cells against x-ray exposure, thereby decreasing the mortality of the mice so exposed.
V. Lackovic et al., (Proceedings of the Society for Experimental Biology and Medicine, 134: 874-879 (1970)) took yeast cell-wall and extracted all constituent matter leaving only "mannans" that he found were responsible for the induction of .alpha.-interferon production by monocytes. The "purified mannans" alleged to be responsible for the physiologic response had a molecular weight of 5,500-20,000 Daltons. He theorized the mannans stimulated mouse peritoneal macrophages to produce the .alpha.-interferon. He does state that his mannans isolated showed no toxicity and "they are poor antigens". There was no mention by Lackovic et al. of the use of these "purified mannans" for antiviral activity or for IL-1 stimulation. We submit Lackovic et al.'s "purified mannans" comprised an assortment of unknown and unidentified substituted and unsubstituted mannans.
Seljelid et al., (Experimental Cell Research, 131: 121-129 (1981)) have observed that insoluble or gel-forming glycans activated macrophages in vitro, whereas the corresponding soluble glycans did not. They postulated that the orientation in which the glycan was presented to the mononuclear phagocyte was decisive for activation. Bogwald et al. (Scandinavian Journal of Immunology, 20: 355-360 (1984)) immobilized glycans that had a stimulatory effect on the macrophages in vitro. This led the authors to believe that the spacial arrangement of the glycan was decisive for the effect on the macrophages in vitro. A purified polysaccharide isolated from Candida albicans induced an antibody response by human peripheral blood lymphocytes in vitro. Wirz et al., Clinical Immunology and Immunopathology, 33: 199-209 (1984). There were significant differences between the anti-Candida antibodies in sera of normal and Candida-infected individuals. Wirz et al., supra.
The antiviral activity of polysaccharides and polysaccharides linked to peptides has been observed. Suzuki et al., Journal of Antibiotics, 32: 1336-134 (1979). Suzuki et al., supra, reported an antiviral action of peptidomannan (KS-2) extracted from culture mycelia of Lentinus edodes. Both oral (PO) and intraperitoneal (IP) administration increased the peak serum interferon titer, which protected mice against viral infections. This was different from dextran phosphate (DP-40) (Suzuki et al., Proceedings of the Society for Experimental Biology and Medicine, 149: 1069-1075 (1975)) and 9-methylstreptimidone (9-MS) (Saito et al., Antimirobial. Agent & Chemotherapy, 10: 14-19 (1976)), which induced higher titers of interferon in mice only if administered intravenously (IV) or intraperitoneally (IP).
Anti-inflammatory activity of Aloe vera gel has been widely reported by both oral testimonies and respected scientific journals. Rubel (Cosmetics and Toiletries, 98: 109-114 (1983)) discussed fully the possible mechanism of the anti-inflammatory effect of aloe gel. Ukai et al., (Journal of Pharmacobio-dynamics, 6: 983-990 (1983)) noted anti-inflammatory activity of polysaccharides extracted from fruit bodies of several fungi. The polysaccharides demonstrated a significant inhibitory effect on carrageenan-induced edema. One of the polymers, O-acetylated-D-mannan (T-2-HN), in addition demonstrated a more marked inhibitory effect than phenylbutazone on scald hyperalgesia. Ukai et al., supra. The assertion that the polysaccharide is free from protein and lipids strongly suggests that the anti-inflammatory effect is due to the acetylated mannan only. Other researchers have also reported anti-inflammatory effects of complex polysaccharides (Saeki et al., Japanese Journal of Pharmacology, 24: 109-118 (1974)), glycoproteins (Arita et al., Journal of Pharmacology., 24: 861-869 (1974)) and sulfated polysaccharides (Rocha E. Silva et al., Biochemical Pharmacology, 18: 1285-1295 (1969)).
Literature reports that polysaccharides possess pharmacological and physiological activities continue to flood the pages of well respected scientific journals. It is therefore not illogical that the mucilaginous gel of the Aloe vera, which is essentially a polysaccharide, holds the secret to Aloe vera's medicinal properties. The discrepancies over whether the polysaccharide is a glucomannan, mannan, pectin or of some other composition are a result of chemical purification steps. By processing aloe according to the present invention, a partially acetylated polymannose has been consistently isolated as the major polysaccharide with pharmacological activity. Yagi et al., (Planta Medica, 31: 17-20 (1977)), using a slightly modified extraction method, isolated acetylated mannan (aloe mannan) from Aloe arborescens Miller var. natalensis. Ovodova (Khim. Prior. Soedin, 83: 93833 (1975)), however, earlier isolated pectin as the main component of the same aloe species.
As discussed above, the biological activity of polysaccharides has been recognized for many years. Polysaccharide materials recovered from plants, yeast and bacteria have demonstrated direct biological activity by eliciting an increase in host defense systems. This reaction is primarily manifested by increased host surveillance for other antigenic substances. Polysaccharides serve as adjuvants (Freund's, etc.) and immunomodulators. They also function as unique T cell-independent antigens. Both cellular and humoral immunity may be affected, and increased phagocytosis of infectious organisms and tumor cells has been observed, as has enhanced production of immunoglobulins.
The structure of these immunologically active polysaccharides and the types of structural variations appear to be the factors that control their potency and toxicity. Their mode(s) of action remain poorly understood; however, recent evidence indicates that several polysaccharides induce lymphocytes and macrophages to produce a wide range of immunologically active substances. The composition of the present invention possesses all of the attributes of these immunologically active substances; it is among the most potent of all known biologically active polysaccharides but differs in that no toxicity has been observed. It also manifests specific antiviral activity through alteration of viral glycoprotein synthesis.